Chemical composition of essential oil and anti trichomonas activity of leaf, stem, and flower of Rheum ribes L. extracts.

OBJECTIVES
Trichomoniasis is one of the most common sexually transmitted diseases in humans and is caused by the protozoan Trichomonas vaginalis. Nowadays, increasing resistance to drugs such as metronidazole resulted in many problem, so new effective remedies are needed. In this study, we evaluate constituents of essential oil and anti-trichomonas activity of Rheum ribes.


MATERIALS AND METHODS
The essential oil from Rheum ribes L. flower growing wild in Iran was analyzed by GC/MS. The parasites were treated with different extract and fractions of the flower, stem, and leave of the plant. Anti-trichomonas activity was evaluated using an in vitro assay.


RESULTS
In all, 19 compounds were identified; palmitic acid [27.08%], n-eicosane [9.9%], n-tetracosane [7.34%], linoleic acid [6.56%], and ethyl linoleate [4.76%] were the main components of the oil. Rheum ribes extracts and fractions concentration dependently inhibited the ability of parasites to growth. This was associated with polarity of solvent used for fractionation and plant parts used for extraction.


CONCLUSION
Findings demonstrate the potential of Rheum ribes extracts as an anti-trichomonas agent for human use. Further studies are required to evaluate its toxicity and safety.


Introduction
Volvovaginitis as a common medical problem can lead to considerable discomfort and repeated medical visits. Infection, allergy, and systemic diseases can happen due to volvovaginitis. Its main causes before menopause are bacterial vaginitis, volvovaginal candidiasis, and Trichomonas vaginalis (Nyirjesy et al., 2006). Trichomoniasis infects more than 100 million new people every year and it is the most prevalent non-viral sexually infection. Infant mortality, preterm delivery or low birth weight, and susceptibility to HIV infection are related to trichomoniasis (Schwebke and Burgess, 2004;Upcroft and Upcroft, 2001;Patel et al., 2000;Petrin et al., 1998).
Metronidazole is the approved drug for the treatment of trichomoniasis, but some resistant strains have been detected (Edwards, 1993;Grossman and Galask, 1990). In addition, it is inhibited in the first trimester of pregnancy, drug allergy, and sometimes for side effects. Therefore, we need to find new alternative drugs for trichomoniasis control. In all over the world, there is a widespread acceptance for using medicinal herbs. Herbal remedies have significant benefits such as fewer side effects, better patient tolerance, and better accessibility (Vermani and Garg, 2002).
Rheum ribes L. is a member of the polygonaceae family which is distributed in Iran and a few neighboring countries (Fazli-bazaz et al., 2005). It is a perennial and cultivated in some temperate countries for its edible red leaf stalks (Dehkardy, 2002;Zargari, 1991). Its Persian name is "Rivas" (Zargari, 1991). Traditionally, it has been used in Iran as sedative and mood enhancer (Sayyah et al., 2009) and in Turkey for the treatment of urinary inflammation and as a diuretic agent (Cakilcioglu and Turkoglu, 2010). In Iran, the plant roots are used as oriental laxative medicine and an anti-psoriatic drug (Shokravi and Agha Nasiri, 1997). Fresh stems and petioles are used as appetizer and digestive in Turkey. Its rhizomes are used to treat hypertension, obesity, and kidney sand and stones (Abu-Irmaileh and Afifi, 2003). Young shoots and petioles are consumed against diarrhea, stomachic, and antiemetic. It is used against hemorrhoids, measles, smallpox, and cholagogue (Baytop, 1999) and also against ulcer, as anti-helmintic, and expectorant (Tabata et al., 1994). The stem and root have been used for the treatment of anemia, anorexia, weakness, anxiety, and it has good effects on major depressive disorder (Sayyah et al., 2009). Its hydroalcoholic extract has some effective in treatment of obsessive compulsive disorders (Sayyah et al., 2011).
In this study, we examined in vitro activity of plant part extracts to figure out which part is more efficiently active against Trichomonas vaginalis.

Plant materials
The fresh leaves, stems and flowers of Rheum ribes in flowering stage were collected in May 2012 from the Alvand mountain, altitude 3111 m, Isfahan province, Iran. The plant was recognized by Mr. Parvazian, technical officer of Faculty of Pharmacy, Isfahan University of Medical Sciences, Iran. The voucher specimen was deposited in the herbarium of faculty of pharmacy and Pharmaceutical Sciences (Voucher specimen No. 2291). Then, it was cut to pieces, air dried for seven days, and were ground into powder.

GC -MS Analysis
The air-dried flower of the plant was powdered. The essential oil of powdered flower was obtained using hydro-distillation. Then essential oil was injected to GC-Mass apparatus (Agilent 6890) equipped with a HP-5MS fused silica column (30×0.25 mm 2 : film thickness 0.25 µm) and interfaced with a Agilent 5975 mass selective detector. The oven temperature was programmed from 60 -280 °C at rate of 4 °C/min. Helium was used as carrier gas at a flow rate of 2 ml/min. Other conditions of the instrument were as follows: ionization voltage 70 eV, injector temperature 280 °C, and ion source temperature 200 °C. Identification of components of oil were based on GC retention indices relative to n-alkenes and computer matching with the WILEY275.L library, as well as by comparison of the fragmentation patterns of the mass spectra with those reported in the literature (Adams, 1995;Davies, 1990).

Extraction and fractionation
The crude extract was prepared by maceration of plant leaves, stems, and flowers powder (200 gr) with methanol (1000 ml) in an air tight container for 7 days at room temperature with occasional shaking. Then, the extracts were filtered through a cotton plug following with a Whatman filter paper. Using a rotatory evaporator, the extracts were concentrated at low temperature and pressure. The crude methanol extracts (10 ml) were reserved for evaluation of anti-trichomonas activity. The remaining extract were mixed with methanol (90 ml) and water (10 ml) and then partitioned using n-hexane (3×100 ml) and dichloromethane (3×100 ml), sequentially. Finally, 11 fractions were obtained including total extract (methanol), hexane fraction, dichloromethane fraction, and residual aqueous fraction. The extracts and fractions were dried in room temperature. The dry extracts were kept in air tight containers for in vitro anti-trichomonas screening (Muhit et al., 2010).

Preparation of test microorganism
Trichomonas vaginalis was obtained from parasitology lab of Isfahan University of Medical Science, which was isolated from vaginal discharge of female patients attending to Obstetric and Gynecology Clinic at Shahrekord. This parasite was cultured in vitro at 37 °C in TYIS33. Log phase culture was diluted with TYIS33 medium for obtaining 10000 cell/ml. Extracts and fractions were diluted with water to obtain final concentration of 0.125, 0.25, 5, and 10 mg/ml and then the extracts and fractions (100 µl) were mixed with parasite (10 µl) + medium (890 µl). Metronidazole (100 µl) + parasite (10 µl) + medium (890 µl) were used as positive control. Medium (100 µl + 890 µl = 990µl) + parasite (10 µl) was used as negative control.
All tubes were incubated at 37 °C. After 24 and 48 hours, samples were taken from each tube and viable parasites, completely active and flagella active were counted with hemocytometer in triplicate. Results of counting are reported as percentage of growth inhibition (G.I %) using following equation: G.I %: = ×100 a: mean number of viable parasites in negative control tube. b: mean number of viable parasites in test tube (Tonkal, 2009;Moon et al., 2006). Table 1 lists retention indices, Kovat indices (KI), and percentage of constituents in the essential oil of Rheum ribes. GC-MS analysis of R. ribes essential oil resulted in the detection of 19 components consisting of 8 alkanes, 5 monoterpenes, 3 fatty acid esters, 2 fatty acids, and 1 sesquiterpene. Anti-trichomonas activity revealed that, no growth was observed after 24 and 48 h of incubation at 1 mg/ml concentration of all extracts and fractions. Subsequently, it was observed that almost all cells were dead at 24 h of incubation at this concentration. However, at lower concentrations of 0.125 mg/ml, the cells were viable even after 48 h of incubation. Viability and number of trichomonas were reduced by several concentrations of extracts and fractions with no live parasites at 0.25 mg/ml for water fraction of flower (Table 2).

Results
With no exception, all the fractions showed greater activity at 48 h than at 24 h probably due to increased contact time of the compounds with the organisms. Although, some fractions were less active than the standard drug metronidazole. 100% of growth inhibition in positive control tube containing metronidazole at first 24 h were observed. The negative control tube showed 0% growth inhibition.
Comparison of anti-trichomonas activity of several fractions (0.25 mg/ml) of R. ribes flower at 24 h is presented in Figure 1.

Statistical analysis
Results are expressed as mean plus/minus standard deviation and the minimal level of significance was considered at p<0.05. All statistical analyses were assessed using SPSS statistical version 17.0 software. Differences among groups were tested by parametric one-way analysis of variance (ANOVA)withTukey'spost-hoc test.
As presented on Table 2, there are fluctuations on anti-trichomonas activity according to plant part extract and solvent used for extract fractionation. Metronidazole has been known as the most effective drug for treatment of Trichomonas vaginalis-related diseases. However, it has been reported that metronidazole has adverse effects and incidence of metronidazole-resistant T. vaginalis has increased. Development of new drugs, which are effective against metronidazole-resistant T. vaginalis has been required.
As presented in Table 2, water fraction of flower of R. ribes is shown to possess the highest percentage of growth inhibition (G.I=100%) with the least concentration (0.5 mg/ml) after 24 h in comparison with positive control. Similarly, 100% of growth inhibition was reported after 24 h of T. vaginalis exposure to Nigella sativa aqueous extract in concentration of 10 mg/ml (Tonkal, 2009). It was reported that 500 µg/ml ethyl acetate extract of Arbutus anedo leaves showed 100% growth inhibition (Ertabaklar et al., 2009). Moreover, the polar compounds including phenolic materials were more effective than semi-polar and non-polar compounds in Eucalyptus camaldulensis (Hassani et al., 2013). It was also shown that other polar compounds such as allicin and ajoene which exist in Allium hirtifolium could exhibit anti-trichomonas activity in comparison with metronidazole (Taran et al., 2006). In addition, researchers reported that berberine isolated from Berberis asisata had in vitro activity compared with metronidazole on T. vaginalis (Soffar et al., 2001). Moreover, alcoholic extracts of Calendula officinalis and Echinacea angustifolia had in vitro efficacy against T. vaginalis (Samochowiec et al., 1979).
Various herbal medications have been used to kill T. vaginalis in vitro. Eighteen aqueous and two ethanol extracts of traditional herbal medicines used to treat trichomoniasis in the Republic of Korea were assessed for their anti-trichomonas activities. Two extracts (Sophorae radix, Phellodendri cortex) showed evident antitrichomonas activity at 8 mg/ml. As presented on Figure 1, anti-trichomonas activity of several fractions of R. ribes flower at concentration of 0.25 mg/ml was varied. The ethyl acetate fraction of Sophorae radix showed anti-trichomonas activity at only 400 µg/ml (Park et al., 2005).
The water fraction of R. ribes flower showed anti-trichomonas activity at 0.125 mg/ml. These findings indicate that R. ribes flower is a potent anti-trichomonas agent. In addition, Kim et al. (2003), Choi et al. (2002), and Park et al. (2004) found that a kalopanax saponin A isolate from Kalopanax pictus and extracts of Sophora flavescens and Gleditsia sinensis have antiprotozoal effects on T. vaginalis by inhibiting cell growth and impairing protein synthesis, respectively. In the aerial parts of R. ribes, chrysophanol, physcion, emodin, quercetin, 5-desoxyquercetin, quercetin 3-O-rhamnoside, quercetin 3-O-galactoside, and quercetin 3-O-rutinoside have been found (Tosun and Akyu¨z-Kızılay, 2003). Ryang et al. (2001) found that extract of Gentiana scabra var buergeri can also inhibit the growth of T. vaginalis. In general, anti trichomonas activity of plant materials seems to depend on solvent and plant used for extraction. Difference in growth inhibition concentration in these plants could be due to different chemical composition.
In conclusion, the present study demonstrated that extracts and fractions obtained from the extracts of different parts of R. ribes exhibit different antitrichomonas activity at several concentrations and can suggest potential use of R. ribes flower water extract for development of an anti-trichomonas drug for human use. Further studies are, however, are required to evaluate its toxicity and safety.